1. Field of the Invention
The present invention correlates to a nucleic acid molecule containing sequence flanking T-DNA insert in transgenic papaya line 18-2-4, which are useful for specifically, reproducibility, sensitively and reliably identifying or reproducing transgenic papaya line resistant to papaya ringspot virus.
2. Description of the Prior Arts
Papaya (Carica papaya L.) is widely grown in tropical and subtropical areas. A destructive disease caused by Papaya ringspot virus (PRSV) is the major obstacle to large-scale commercial production of papaya throughout the world (Purcifull et al., 1984, CMI/AAB Descriptions of Plant Viruses. No. 292). PRSV was first found in southern areas of Taiwan in 1975 (Wang et al., 1978, Plant Prot Bull 20:133-140). Since then it has destroyed most of the papaya production in commercial orchards. PRSV is a member of the genus Potyvirus (Fauquet et al., 2005, Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. ELSEVIER, San Diego), the largest and economically most important plant virus group. The virus is naturally transmitted by aphids in a non-persistent manner and induces symptoms of mosaic and distortion on leaves, streaks on petiole and stem, and stunting in growth, resulting in drastic reduction in fruit quality and yield (Purcifull et al., 1984, supra).
Several control measures have been used to protect papaya plants from PRSV infection, including the selection of planting time to avoid the peak of winged aphids, the use of silver mulch to repel aphids from visiting seedlings, and the application of cross-protection using mild strains of PRSV (Yeh et al., 1988, Plant Dis 72:375-380; Yeh and Gonsalves, 1994, Adv Dis Vector Res. 10:237-257). However, none of these methods provides a long period of effective protection against PRSV. Currently, cultivation under netting to prevent papaya plants from infection by aphid-transmitted PRSV has become an effective control method in Taiwan. However, high cost of the netting, creation of environmental hazard owing to the difficult degradation of plastic material in nature, and high risk of destruction by tropical storms are major concerns (Bau et al., 2003, Phytopathology 93:112-120).
In recent years, based on the concept of pathogen-derived resistance (PDR) (Sanford and Johnson, 1985, J Theor Biol 113:395-405), the development of transgenic plants containing a genomic segment of a plant virus is widely used as a strategy to control corresponding plant viruses (Beachy, 1997, Curr Opin Biotech 8:215-220). In most cases, the mechanism of resistance occurs posttranscriptionally, by a RNA-mediated process that targets both the viral RNA and transgenic mRNA for degradation in a sequence-specific manner (English et al., 1996, Plant Cell 8:179-188; Lindbo et al., 1993, Plant Cell 5:1749-1759; Sijen and Kooter, 2000, Bioessays. 22:520-531; Vaucheret et al., 1998, Plant J 16:651-659). The coat protein (CP) gene of PRSV has been transferred into papaya via particle bombardment (Fitch et al., 1990, Plant Cell Rep 9:189-194) and transgenic lines highly resistant to PRSV infection have been selected (Fitch et al., 1992, Bio/Technology 10:1466-1472; Lius et al., 1997, Mol. Breed. 3:161-168).
Transgenic papaya lines have been successfully commercialized in Hawaii since 1998 (Gonsalves, 2002, Curr Top Microbiol Immunol 266:73-83; Tripathi et al., 2007, Methods Mol Biol 354:197-240). In Taiwan, transgenic papaya lines carrying the CP gene of a Taiwan severe strain PRSV YK, have also been generated by Agrobacterium-mediated transformation (Cheng et al., 1996, Plant Cell Rep 16:127-132). When the transgenic papaya lines are challenged with PRSV YK, their reactions to virus infection range from high susceptibility to complete resistance (Bau et al., 2003, Phytopathology 93:112-120). The transgenic lines 16-0-1, 17-0-1, 17-0-5 and 18-2-4 provide broad-spectrum resistance against PRSV strains from different geographic origins under greenhouse conditions (Bau et. al., 2003, supra) and high degrees of resistance during field tests (Bau et al., 2004, Plant Dis 88:594-599). These lines have great potential for control of PRSV in different geographic areas.
It was observed that the highly resistant lines 16-0-1, 17-0-5 and 18-2-4 display a strong strain-specific resistance at young development stage (5-cm height), but not other lines, such as 18-0-9 and 19-0-1. Accordingly, the insertion locus of T-DNA in papaya genome must have its unique characteristics, particular to the papaya line 18-2-4. Once the locus of the genome inserted by the T-DNA is elucidated, it will be helpful for generating a stable papaya line with broad spectrum of resistance against PRSV strains since people can utilize the character of the sequence of T-DNA insert in combination with genome sequences flanking the T-DNA to modify various papaya strains to obtain resistances to PRSV by homologous recombination without tedious selections and field tests.
On the other hand, concerns have recently been raised on safety issues of genetically modified organisms (GMOs) (Singh et al., 2006, Appl Microbiol Biotechnol 71: 598-607). Variegated legislations have been applied in different countries for the GM food approval and labeling. According to European Union (EU) regulation (EC) No. 1830/2003, it is required to be able to trace GMO and products derived from GMOs at every stage on market from production to distribution (European Parliament and Council of European Union, 2003, p. 1-5). Also, regulations in Taiwan for GM crop field tests and consequent variety rights also requires the information of genomic sequences flanking the transgene for a particular transgenic line. Therefore, characterization of a particular GMO to provide specific and reliable methods for even-specific detection is crucial to assure regulatory compliance and also important to monitor unauthorized GMO occurrence in markets and plantations.
Accordingly, there is an urgent need for means for generating or identifying transgenic papaya lines with broad-spectrum of resistance against various PRSV strains, as transgenic papaya line 18-2-4, which is specific, reproducible, sensitive and reliable. To overcome the shortcomings, the present invention tends to provide isolated nucleic acids, methods, primers, probes and kit for generating or identifying transgenic papaya line 18-2-4 to mitigate or obviate the aforementioned problems.